A fundamental question concerning spontaneous organ specific autoimmune disorders is whether there exist essential target autoantigens such that deletion of an immune response to one or more of such targets will abrogate disease. During the past 2.5 years with support from the current grant we have produced NOD mice lacking both the native insulin 1 gene, native insulin 2 gene, and mice lacking both insulin 1 and insulin 2 genes rescued from diabetes with a transgenic preproinsulin gene mutated to have alariine at position B16 rather than tyrosine of the B:9-23 sequence(RIP[Rat Insulin Promoter]-B16:A preproinsulin). In addition we have produced T Cell Receptor Transgenic NOD mice with the BDC 12-4.1 anti-B:9-23 insulin peptide T cell receptor. The knockout of the insulin 2 gene increases insulin autoantibodies and accelerates the development of diabetes; knockout of the insulin 1 gene prevents 90% of the progression to diabetes but insulitis remains in the majority of mice and insulin autoantibodies are unchanged;and finally we have our first mice with the double knockout and preliminary data that anti-insulin autoantibodies, insulitis and diabetes is prevented. The latter is a remarkable result, suggesting but not proving that the native insulin B:9-23 sequence is essential for anti-islet autoimmunity of the NOD mouse. Given the dramatic effect on disease with multiple transgenic founders with the BT6:Alanine preproinsulin combined with dual insulin gene knockouts (insulin 1 and insulin 2) it is imperative to directly demonstrate effects of the transgene sequence and to define the etiology of protection in the absence of native insulin B:9-23 sequences. We believe this will provide critical information and knowledge relative to immunopathogenesis as well as a model for deletional therapy in a complex spontaneous autoimmune disorder. We propose to: 1. Analyze additional NOD mice carrying both native insulin 1 and 2 knockouts with B16 alanine mutated insulin for expression of insulin autoantibodies, insulitis and development of diabetes. 2. Produce insulin 1 and 2 knockout NOD mice with a tetracycline regulated transgene with native insulin 1 and insulin 2 sequence and analyze these mice for restoration of anti-islet autoimmunity. 3. Evaluate the mechanism of protection in terms of autoreactive pathogenic and regulatory T lymphocytes. 4. Analyze autoreactive T lymphocytes(both CD4 and CDS) targeting multiple epitopes of insulin and multiple antigens of the above NOD mice, including analysis of whether islets of double knockout B16:A mutated insulin are resistant to recurrent islet autoimmunity. The long-term vision for this pathway of research is to understand enough concerning autoimmunity to an essential target peptide of insulin in a spontaneous complex disease model to develop strategies to monitor and prevent type 1A diabetes using conceptually similar strategies with either the same or other insulin determinants.